Fasciola hepatica: Isolation and characterisation of a cathepsin L proteinase. Thesis Presented for the Degree of DOCTOR OF PHILOSOPHY by Angela M. Smith, B.Sc. under the supervision of John P. Dalton, Ph.D. School of Biological Sciences Dublin City University. I hereby certify that this material, which I now submit for assessment on the programme of study leading to the award of Ph.D. is entirely my own work and has not been taken from the work of others save and to the extent that such work has been cited and acknowledged within the text of my work. Signed: l/v Date Angela 1VI. Smith Date:. ACKNOWLEDGEMENTS I would like to thank Dr. John Dalton for all he has done for me during the last four years. His help, guidance and enthusiasm were much appreciated. I would also like to thank Dr. Paul Brindley for his help and advice during my six month visit to his laboratory in Australia, Dr. Alan Trudgett and his colleagues in Queen's University Belfast for their collaboration in the immunolocalisation studies, and Dr. Carlos Carmona of the Universidad de la República, Montevideo, Uruguay for his contribution to the antibody inhibition studies during his visit to our laboratory in Dublin. I would also like to express my gratitude to my parents for giving me so many opportunities, and to all my friends for their support and encouragement, especially during the last six months. Finally, I wish to thank the members of the Parasitology lab. in D. C. U. for the good times and the never boring lab excursions! To my parents. CONTENTS Abstract 1 Abbreviations 2 1.0 Introduction 4 2.0 Materials and Methods 36 2.1 Materials 37 2.2 Methods 41 2.2.1 Preparation of in vitro-released products from adult Fasciola hepatica 41 2.2.2 Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) 41 2.2.3 Gelatin-substrate gel analysis of fluke in vitro released products 42 2.2.4 Protein estimation 42 2.2.5 HPLC analysis of E/S products 43 2.2.6 Assay for lgG2a cleaving activity 43 2.2.7 Proteinase assays with synthetic fluorogenic peptide substrates 43 2.2.8 Fluorogenic visualisation of proteinases in SDS-PAGE 44 2.2.9 Inhibition studies using diethylpyrocarbonate (DPC) and Z-F-A-CHN2 44 2.2.10 Purification of F. hepatica\gG cleaving cysteine proteinase 45 2.2.11 N-terminal sequence determination 46 2.2.12 Production of a polyclonal antiserum 46 2.2.13 Immunoblotting 47 2.2.14 Immunolocalisation studies 47 2.2.15 Inhibition of proteinase activity using anti- cathepsin L-like proteinase antibody 48 2.2.16 RNA isolation from adult F. hepatica worms 49 2.2.17 mRNA isolation 50 2.2.18 cDNA preparation 51 2.2.19 Construction of oligonucleotide primers 53 2.2.20 Polymerase chain reaction (PCR) 55 2.2.21 Subcloning of PCR gene fragments 55 2.2.22 Screening of recombinant colonies 56 2.2.23 Sequencing of subcloned PCR gene fragments 57 3.0 Results 58 3.1 Characterisation of IgG cleaving enzyme in adult fluke E/S products 59 3.1.1 Demonstration of IgG cleavage 59 3.1.2 HPLC analysis of E/S products 61 3.1.3 Direct visualisation of proteinases in HPLC fractions 61 3.2 Inhibition studies with DPC and Z-F-A-CHN2 64 3.2.1 Inhibition of the active site histidine residue with DPC 64 3.2.2 Inhibition with Z-F-A-CHN2 67 3.3 Purification of the cathepsin L-like cysteine proteinase 67 3.4 N-terminal sequence determination 69 3.5 Immunoblotting studies 74 3.6 Light- and electron-microscope immunolocalisation studies 78 3.7 Inhibition of proteinase activity with anti-cathepsin L-like proteinase antibodies 81 3.7.1 Inhibition of GS-PAGE proteolytic activity 81 3.7.2 Inactivation of the IgG cleaving ability of the proteinase 84 3.7.3 Antibody-mediated eosinophil attachment to juvenile flukes 84 3.8 Cloning and sequencing of PCR amplified cysteine proteinase gene fragments 85 3.8.1 PCR amplification of cysteine proteinase gene fragments 86 3.8.2 Subcloning and sequence analysis 86 4.0 Discussion 93 5.0 References 122 6.0 Appendix 147 ABSTRACT Fasciola hepatica, a parasitic trematode, is the causative agent of liver fluke disease. It has been shown previously, that both the migratory and adult worm stage of the parasite secrete multiple cysteine proteinases when they are cultured overnight (Dalton & Heffernan, 1989). In this study, one of these proteinases has been purified by standard chromatographic techniques. The purified enzyme was characterised as a cathepsin L-like proteinase using synthetic substrates, inhibition studies, N-terminal sequencing and immunolocalisation studies. This is the first cathepsin L-like proteinase to be identified in a parasitic trematode. This cathepsin L-like proteinase is capable of cleaving immunoglobulin molecules, and is able to protect newly excysted juveniles from destruction by immune-effector cells when it is included in an eosinophil adherence assay. Antibodies to the purified proteinase are able to neutralise its proteolytic activity in vitro. A partial gene fragment encoding the cathepsin L-like proteinase has been obtained using PCR and subcloning techniques. The cathepsin L-like proteinase is present in all stages of F. hepatica and, hence, is considered an ideal target molecule at which to design a vaccine and/or drug, for use in the control of this agriculturally important parasitic disease. 1 ABBREVIATIONS BCIP 5-bromo-5-chloro-3-indolyl phosphate Bisacrylamide N, A/“-Methylene bisacrylamide BSA Bovine serum albumin DMSO Dimethyl sulphoxide DPC Diethylpyrocarbonate DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid disodium salt E-64 fra/7s-epoxysuccmyl-L-leucylamido(4-guanidino) butane FCS Foetal calf serum FITC Fluorescein isothiocyanate Hepes N-[2-hydroxyethyl] piperazine-N’[2-ethane sulphonic acid] IPTG Isopropyl-B-thiogalactopyranoside NBT Nitro blue tétrazolium PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline PMSF Phenylmethylsulphonyl fluoride RPMI Roswell Park Memorial Institute SDS Sodium dodecyl sulphate TEMED N, N, N’, N’-tetramethylethylenediamine Tris tris-(hydroxymethyl)-methylamine (2-amino- hydroxylmethyl) propane-1,3-diol Z-F-A-CHN2 /V-benzyloxcarbonyl-L-phenylalanine-L-alanine- 2 diazomethylketone Z-F-R-AMC A/-benzyloxcarbonyl-L-phenylalanine-L-arginine-7- amino-4-methylcoumarîn.HCI Z-R-AMC /V-benzyloxcarbonyl-L-arginine-7-amino-4- methylcoumarin.HCI Z-R-R-AMC A/-benzyloxcarbonyl-L-arginine-L-arginine-7-amino- 4-methylcoumarin.HCI 3 CHAPTER ONE INTRODUCTION 4 1.0 INTRODUCTION In 1947, Professor Stoll drew attention to the worldwide presence of helminth parasites in his article “This wormy world”. Helminth parasites infected 70 % of the then world population of approximately 2 billion (Stoll, 1947). Since that time, the prevalence of helminth infections has kept pace with the growth of the world population. If the trend continues till the year 2100, a predicted world population of 7-15 billion would harbour 5-10 billion helminth infections, unless special control measures are undertaken (Crompton, 1987). The term “helminth” (derived from the Greek words helm ins or helminthos), literally means “worm”, zoologically speaking however, it has a more precise connotation and is currently restricted to members of the phyla Platyhelminthes, Nematoda and Acanthocephala (Smyth, 1976). The study of helminths is now regarded as being confined to the study of parasitic worms. Helminths typically parasitise vertebrates, although invertebrates act as intermediate hosts. The helminth diseases in man and domestic animals are caused by three groups of parasites belonging to the classes of trem atoda (flatworms), nematoda (roundworms), and cestoda (tapeworms), and are distributed throughout the world (Singh & Sharma, 1991). There are approximately 200 recognised helminth parasites of man. Table 1.1 lists the parasites which are most common in humans. For most helminth infections the relationship between between infection and disease is complex, and disease is not necessarily an automatic outcome of infection (Bundy etal., 1992). Only a small proportion of those individuals with heavy infections are likely to develop overt disease. There is a low mortality/high morbidity rate associated with helminth infections, so although 5 Table 1.1 Parasitic helminth infections which are common to man, an example of a causative agent of each infection, and the numbers infected. Data obtained from Hopkins, (1992). Parasite infection & example Millions infected Ascariasis (Ascaris lumbricoides) 1000 Hookworm (Ancylostoma duodenale) 900 Trichuriasis (Trichuris trichiura) 750 Schistosomiasis (Schistosoma mansoni) 250 Filariasis (Wuchereria bancrofti) 90 Taeniasis (Taenia saginata) 70 Onchocerciasis (Onchocerca volvulus) 30 Fascioliasis (Fasciola hepatica) 17 Trichinosis (Trichinella spiralis) 11 millions of people may be infected with helminths, relatively few will actually die as a result of infection, which seems to prevent well-focused investigation into their control and treatment (Parkhouse & Harrison, 1989). In fact it is estimated that at least one quarter of the worlds population is infected with helminthic parasites (Bundy, 1992), and about 150,000 die each year as a result of these infections (Bundy, 1990). One feature in the evolution of some animals is the increasing complexity of their alimentary, respiratory and circulatory systems. The development of such systems was, of course, advantageous to these evolving organisms, but it was not without some inherent disadvantages. As each new organ system evolved, 6 especially those containing cavities or surfaces, it presented a habitat for potential parasites. These cavity containing organs appeared especially in vertebrates and every part of the vertebrate body capable of supporting parasitic life has been invaded (Smyth, 1976). A majority of helminths use the gastrointestinal tract as their favourite niche; however some parasites may also invade musculature, the blood circulatory system, and other parts of the body such as lungs, liver, lymphatics, and eyes, producing serious clinical complications.